Actin Assay

Fig. 8.9 (a) Western blot of C-Kit inhibition. WBZ 4 inhibits phosphorylation of C-Kit kinase in ST-882 GIST cells. Gel bands from the western blot assays of C-Kit and its phosphorylated (P) form in GIST cells treated with WBZ 4 and imatinib. The fi-actin assay was adopted as control, (b) Western blot of Bcr-Abl inhibition. Phosphorylation of Bcr-Abl kinase is not significantly inhibited by WBZ 4 in K562 CML cells. Electrophoretic gel bands for western blots for Bcr-Abl kinase and its phosphorylated (P) form in CML cells treated with WBZ 4 and imatinib. Reprinted from the [14], copyright 2007 with permission from the American Society for Clinical Investigation... 

In general, the two motility assays give the same values for velocity and have similar characteristics. In each case the movement is assumed to be "unloaded" since the velocity does not depend on the concentration of myosin bound to the bead or to the glass surface above a certain threshold level (Collins et al., 1990 Sellers et al., 1985 Sheetz et al., 1984). The velocity is also not dependent on the length of actin filaments in the sliding actin assay (Collins et al, 1990 Warshaw et al., 1990). In both systems the direction of movement is determined by the polarity of actin (Sellers and Ka-... 

Fig. 2. Comparison of the pyrenyi-actin assay and in vitro TiRF microscopy as toois for the anaiysis of actin assembiy. Actin-binding proteins have different effects on actin poiymerization. Whereas the pyrenyi-actin assay does not aiiow to cieariy distinguish between de novo nucieation (e.g., by formins), nucieation through branching on preexisting fiiaments (by Arp2/3-compiex) or enhanced fiiament eiongation (e.g., by VASP), these events can be unambiguousiy discriminated by in vitro TiRF microscopy.

Figure 13, A schematic diagram of the motility assay. Myosin molecules (HMM or S-1 are also used) stick to glass coverslips coated with nitrocellulose. Actin, in solution, is then added to the glass coverslip and it binds to the myosin molecules. When ATP is added, actin can move over the surface, propelled by the myosin molecules.

Leira, F., et al., Development of a F actin-based live-cell fluorimetric microplate assay for diarrheic shellfish toxins. Anal. Biochem., 317, 2, 129, 2003. 

Molecules (chemoattractants) that stimulate neutrophil-directed migration (chemotaxis) bind to distinct receptors on neutrophil plasma membranes (discussed in Chapter 38 of this text). Within seconds after chemoattractant binding, neutrophils exhibit rapid oscillations in actin polymerization and depolymerization (12,13). The shape changes accompanying chemoattractant binding depend on the duration and extent of F-actin polymerization (3). These quantitative studies of F-actin content were performed utilizing a flow cytometric assay that detects the fluorescence intensity of individual, fixed, permeabihzed cells that have been stained with F-actin-specific, fluorescent phallotoxins (14,15). 

The phallotoxins, phalloidin and phallacidin, are bicyclic peptides with mol wts of 789 and 847 Dalton, respectively. The NBD phallacidin and rhodamine phalloidin conjugates have been most frequently utilized in flow cytometric assays. The fluorescent phallotoxin conjugates have mol wts of 1000-1200 Dalton, are water-soluble, and stain actin at nanomolar concentrations (reviewed in ref. 19). Unconjugated phallotoxins should be obtained to verify the specificity of fluorescent phallotoxin staining to F-actin (see Note 4). 

Although this chapter has focused on neutrophil activation events associated with F-actin changes, this assay has also been applied to studies of lymphocyte and platelet activation (20-22). 

The work that follows pertains primarily to actin networks. Many proteins within a cell are known to associate with actin. Among these are molecules which can initiate or terminate polymerization, intercalate with and cut chains, crosslink or bundle filaments, or induce network contraction (i.e., myosin) (A,11,12). The central concern of this paper is an exploration of the way that such molecular species interact to form complex networks. Ultimately we wish to elucidate the biophysical linkages between molecular properties and cellular function (like locomotion and shape differentiation) in which cytoskeletal structures are essential attributes. Here, however, we examine the iri vitro formation of cytoplasmic gels, with an emphasis on delineating quantitative assays for network constituents. Specific attention is given to gel volume assays, determinations of gelation times, and elasticity measurements. 

Figure 3. Critical concentration behavior of actin self-assembly. For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by methods that measure the sum of addition and release processes occurring at both ends. Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscosity measurements. Forthe bottom curves, the polymerization behavior is typically determined by fluorescence assays conducted under conditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1 -actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs). Note further that the barbed end (or (+)-end) has a lower critical concentration than the pointed end (or (-)-end). This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition to the (+)-end at the expense of the subunit loss from the (-)-end.

Actin filaments affinity column [preparation, 196, 49 properties, 196, 305] assay [in cells, 196, 486 in platelet lysates, 215, 54] associated proteins, identification, 215, 50 attachment to col-... 

Actin-binding proteins ABP-50, purification, 196, 78 ABP-120, purification, 196, 79 ABP-240 purification, 196, 76 effect on actin depolymerization, 215, 74 extraction, 196, 311 isolation from Dictyostelium discoideum, 196, 70 platelet-derived actin binding proteins [characterization, 215, 58 purification, 215, 58, 64 recombination with actin, 215, 73] 30-kDa Dictyostelium discoideum actin-crosslinking protein [assays, 196, 91 preparation, 196, 84] actin-depolymerizing factor [assay, 196, 132[ DNase assay, 196, 136 platelet-derived a-actinin [characterization, 215, 58 purification, 215, 58, 70 recombination with actin, 215, 73]. 

ACTIN-BASED BACTERIAL MOTILITY ACTIN ASSEMBLY ASSAYS ACTIN ASSEMBLY KINETICS ACTIN FILAMENT CAPPING PROTEIN ACTIN FILAMENT SEVERING PROTEIN... 

ACTIN POLYMERIZATION ASSAYS FARADAY S CONSTANT NERNST EQUATION FARADAY S LAWS... 

ACTIN ASSEMBLY ASSAYS MINI-CHAPERONES Mini-chromatography column,... 

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